Rapid cell block embedding method and apparatus

ABSTRACT

A method and apparatus for embedding cells that utilizes a flow-through embedding technique maximizes the efficiency of extractions and decreases time for embedding the cell fragments, minimizes cell loss, and automatically positions cell samples at the position in which a microtome blade will section them. The apparatus includes a cell flow pathway defined by an inflow tube for delivering cell fragments from a cell sample to a sample port. The sample port is in fluid communication with a tissue cassette having attached thereto a filter. The cell flow pathway is in communication with a reagent flow pathway for delivering the reagents through the sample port to the cassette. The apparatus is configured such that the application of pressure directs the cell fragments from the cell sample through the cell flow pathway, and effects delivery of the reagents through the reagent flow pathway. The apparatus produces an embedded cell block having concentrated cells near the plane of the block to be sectioned in a quick and efficient manner.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on U.S. provisional application No. 60/422,768entitled “Automated Cell Block Embedding Apparatus” and filed on Oct.31, 2002, of which priority is claimed.

FIELD OF THE INVENTION

The present invention relates to methods and devices for preparing cellsfor microscopic examination, and for archiving the cells for molecularand immunologic tests. More particularly, the invention relates to amethod and apparatus for embedding cells and tissue fragments within asolid substrate, such as paraffin, to enable them to be cut with amicrotome for microscopic evaluation, and to be stored in a stablestate.

BACKGROUND

Many disease processes can only be diagnosed on the basis of histologicor cytologic examination using a light microscope. For instance, whilethe presence of a tumor can be detected using radiological devices, thedetermination of whether a tumor is benign or malignant still requires apathologist's interpretation of the appearance of the cells using lightmicroscopy. Before reaching this stage, however, the tissue sample mustfirst be retrieved, collected, and processed for microscopicexamination. A number of techniques are available for retrieving andcollecting biopsies or cell samples from a patient. It is of benefit topatients to use minimally invasive techniques for obtaining biopsies orcell samples. For example, small tissue fragments can be obtained fromfine needle aspiration biopsy, or by brushing body cavity surfacesaccessible through minimally invasive endoscopic techniques. Onceretrieved, the cells then need to be processed for microscopy. A varietyof processing techniques are known, including the cytospin® techniqueand the thin-prep® technique for depositing tissue fragments directlyonto a microscope slide.

Another technique, commonly referred to as a cell block preparation, hasseveral advantages over the direct deposition of tissue fragments. Thecell block procedure immobilizes cells or small tissue fragments in asolid support, typically paraffin wax. Thin sections of the cell blockare then cut with a microtome and the sections mounted onto a microscopeslide for examination. The resulting sections from the cell blockdisplay diagnostic information in a manner that complements the directdeposition techniques. For example, the architectural arrangements ofcells to each other is displayed better in section from a cell blockthan in directly deposited cells on a microscope slide. Cell blocks alsopermit important diagnostic molecular and immunological tests to beconducted on the cell samples that would otherwise be difficult orimpractical on direct preparations. In addition, cell blocks appear topreserve the cells indefinitely in a convenient manner at roomtemperature, thereby facilitating biomedical research.

The cell block preparation method requires that the cell fragments be“embedded” in a solid medium, most commonly paraffin wax. “Embedding”requires the following generic steps: (1) all water molecules must beremoved from the cells, typically by alcohol (water is miscible withalcohol); (2) all alcohol must then be removed, as well as all fattysubstances, and replaced typically by xylene (xylene is miscible withalcohol but not water); (3) the xylene must be removed and replaced withwax (wax is miscible with xylene but not with most alcohols or water);and (4) the cells in molten wax must then be manually organized andhardened on the underside of a tissue cassette so that a section of thewax block with the embedded tissue can be cut using a microtome. Thefirst three of these steps are commonly performed by a “tissueprocessor,” a machine that circulates alcohol, xylene, and molten waxsequentially in a chamber containing the tissue cassette. Tissuecassettes typically serve the dual purpose of containing the cell sampleduring the embedding process, and for providing an attachment mechanismfor holding the wax block on the microtome machine so that the cellsample subsequently embedded in wax on the undersurface of the cassetteis able to be cut into thin sections.

Before “embedding” can take place, the cell sample must be manipulatedto concentrate the cells and facilitate their transfer through theembedding procedure. A commonly used procedure for preparing such cellblocks is the clot technique, which is described by Yang et al. in ActaCytologica, 42:703-706 (1998). The clot technique involves the followinggeneric steps: (1) a cell sample is centrifuged for 10 minutes; (2) thesupernatant is manually poured off, leaving a concentrated cell button;(3) fibrin and thrombin, obtained from blood bank supplies, is manuallyadded to the cell button and incubated for 15 minutes with occasionalmanual swirling to trap the cell button into the clotting fibrin; (4)the clotted cell sample is removed manually from the centrifuged tubewith care to avoid cell loss due to streaking the clot along the side ofthe tube or breaking the clot into impractically small pieces; (5) theclotted cell sample is manually transferred to lens paper, which is thenfolded over and placed manually into a tissue cassette; (6) the tissuecassette is then manually placed into an automated tissue processor,which then cycles alcohol, xylene and hot paraffin into the machine (asdescribed in the preceding paragraph) and is typically set to operateovernight; (7) the following morning, the cassette is manually removedfrom the liquid paraffin of the tissue processor and opened; (8) thelens paper is opened, and the cell clot is scraped off the lens paperand manually placed into a tissue section mold; and (9) paraffin isgently added to the mold while manually trying to maintain the cell clotagainst the lowest surface of the mold that will eventually be cut. (10)The tissue cassette is then inverted over the mold to serve as a holderfor the microtome machine, and hardened into the wax along with the cellclot. (11) The tissue cassette, with included wax-embedded cellfragments, is then separated from the mold. At this point, the wax blockis ready to be cut with a microtome. Many of these eleven steps arecommon to other existing cell block production techniques.

Another popular procedure for preparing cell blocks is the collodion bagtechnique, which is described by Fahey and Bedrossian in LaboratoryMedicine, 74(2):94-96 (1993). The collodion bag technique involves allof the eleven steps above with the exception that steps 1-4 are replacedby the following: Collodion is manually poured into a centrifuge tube tocoat the tube. The cell sample obtained from the patient is thencentrifuged in the coated tube. The supernatant is poured off, and thethin coating of collodion is pulled from the tube with the includedconcentrated cell button and embedded as in steps 5-11 above. Thecollodion technique provides an advantage over the clot technique byavoiding dilution of the cells with fibrin and thrombin. With thecollodion technique, no waiting is required for the cell clot to form asis required of the clot technique, and cells are not susceptible tobeing lost as they are pulled out of the centrifuge tube. However, thecollodion technique is substantially more dangerous than the clottechnique due to the flammable nature of collodion and its ethersolvent.

Yet another technique for preparing cell blocks is described byDiaz-Rosario and Kabawat in Cancer, 90:265-272 (2000). In thistechnique, the cell sample is initially filtered and the filtered sampleis scraped from the filter and transferred onto lens paper which is thenfolded and placed into a tissue cassette and transferred to a tissueprocessor, followed by steps 6-11 above.

Currently available cell block preparation techniques such as the onespreviously described suffer from a number of problems that make themcumbersome, costly, susceptible to contamination or mislabeling, andinefficient for showing diagnostic cells in the final microtomesections. For instance, many of the techniques require that thewax-embedded cell fragments be manually transferred to a tissue cassetterequired to hold the wax block onto the microtome for sectioning. Thismanual transfer to the tissue cassette takes a considerable amount oftime, as does the step of transferring the cells from a sample tube intoa tissue processor. Many of the cell fragments are wasted during thetransfer and/or embedding steps. In the past, techniques attempting toimprove the concentration of cells within the sectionable material andavoid cell waste have proven less than ideal, because the techniquesnecessarily involve dilution of the microtome section with carriersubstances such that relatively few cells are present, or do notdecrease cell loss in pre-embedding steps.

Current cell block techniques typically take anywhere from 8 to 16 hoursto complete because the extraction of water, alcohol, fats, and xylenein the tissue processor is time consuming. While there are methods tospeed up the tissue processing step (step 6 above), such as by usingmicrowave radiation, vacuum pressure, elevated temperatures, and morerapidly diffusing chemicals, these methods suffer from their own set ofproblems. These techniques only modestly decrease the tissue processingtime (step 6 above) but are relatively tedious to establish in thelaboratory. Furthermore, these techniques do not enhance the efficiencyof the other processing steps. The ability to produce same-day cellblocks would enable faster diagnoses to be rendered, with cost savingsamplified throughout the health care system.

Another drawback with the cell block techniques described above are thatthey are also susceptible to mislabeling of a sample since the samplehas to be manually moved between the sample tube, lens paper (orcollodion, and other carrier substances), tissue cassette, and tissuemold. In addition to a susceptibility to complete mislabeling, crosscontamination between the patient's sample and cells and biomolecules(including cancer cells) of other sources is also possible with existingtechniques. That is, in the fibrin clot technique, the patient's cellsare mixed with pooled plasma from other patients in order to form aclot. During transfer of cells to and from the tissue cassette,contamination of cells from one case to another can occur on the forcepsused to manually handle the cell buttons. Since multiple tissuecassettes are simultaneously immersed in a common bath of reagents intissue processors, the possibility for cross contamination of cancercells from one patient to the cell button of another sample (referred toby pathologists as “floaters”) is a well-recognized serious problem.

Yet another disadvantage of current tissue processor steps is that thesesteps are difficult to standardize. This is because one tissueprocessing machine typically circulates the embedding reagents for manylaboratory samples at one time. A cell block ready to be placed on atissue processor at 9 AM would necessarily be exposed to differentconditions from a sample placed on the same processor at 9:30 AM.Emerging molecular techniques require standardized processing foroptimal performance.

Since light microscopy is currently the “gold standard” for diagnosis ofmany diseases, advances in understanding of the molecular biology ofdiseases and their treatments generally requires the ability tosimultaneously study the microscopic features of cells while preservingtheir constituent molecules for research. Simultaneous preservation ofmorphology and the biochemical constituents of a cell currently pose anumber of problems for researchers and diagnostic pathologists (reviewedin Srinivasan M, Sedmak D, and Jewell S. “Effect of fixatives and tissueprocessing on the content and integrity of nucleic acids ”. Am J Pathol.2002; 161:1961-71.) Freezing tissues preserves nucleic acids andproteins (though some autolysis is inevitable during the thawing of thetissue), but frozen tissue presents obstacles to morphologic assessment.Some of the obstacles include “freezing artifacts” that distortmorphology and hinder classical light microscopy diagnosis, technicaldifficulties in cutting thin sections of frozen tissue, inability to cuta histologic section from certain types of frozen tissues (for exampletissues containing abundant fats), cumbersome and expensive storage offrozen tissue samples, impracticality of freezing very small tissuesamples, and the expense of special microtomes needed to cut frozentissues. Paraffin-embedded cell samples provide excellent morphology andbiomolecules such as nucleic acids and proteins appear very stable forlong term storage even at room temperature, if the biomolecules survivethe embedding steps. Unfortunately, formaldehyde fixation, widely usedas a fixative in production of a paraffin embedded tissue block, causesextensive damage to nucleic acids and proteins. While there are paraffinembedding techniques that do not use formaldehyde, these techniquesstill pose problems. The problems include the slow rate of diffusion ofthe embedding reagents which permits degradation of biomolecules, day today variation in fixation conditions when a bulk embedding reagent isused over many days to embed samples, and the potential forcontamination of cells from one sample into another as samples arebathed together in vats of the reagents in existing paraffin embeddingtechniques.

There is thus a need for a cell block preparation technique that is ableto place cell fragments within a concentrated area within the cellblock, without losing cells, such that a single section of the wax cellblock shows a substantial proportion of all the cell fragments withoutdilution by fibrin or other carrier molecules. Also desirable is atechnique which avoids or eliminates mislabeling of a cell block,contamination of one cell block with another cell block, and whichrequires less embedding reagents and time than currently availabletechniques, and allows standardized preservation of biomolecules fordiagnostic studies and research.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus that utilizes aflow-through processing and embedding technique for rapidly embeddingcells. This flow-through processing maximizes the efficiency of cellrecovery and of extractions during embedding, thereby decreasing theamount of cellular sample required, minimizing the amount of time forprocessing, and minimizing the amount of reagents needed for embedding.The method and apparatus also automatically places cells at the plane inthe cell block where they need to be sectioned without diluting themwith carrier substances. In addition, the method and apparatus minimizesor eliminates the potential for mislabeling of a cell block or crosscontamination between two different cell samples. Finally, the methodallows customization and standardization of cell preservation or cellembedding conditions to facilitate cell research.

According to an exemplary embodiment of the present invention, theapparatus includes a modified tissue cassette that serves a dualfunction of capturing the cell sample and also providing a pathwaythrough which the embedding reagents can flow. A cell flow pathway isprovided for delivering cell fragments from a collected cell sample,typically in either an aqueous solution or a liquid cell-preservative,through the modified tissue cassette to a filter that traps the cellsand tissue fragments. The apparatus also includes a reagent flow pathwayconfigured to sequentially pass embedding reagents, then lastlyparaffin, through the tissue cassette and through the cell sampledeposited on the filter. When the paraffin is cooled, the filter ispeeled away from the paraffin embedded cells, and a gasket between thefilter and the tissue cassette is pulled away from the tissue cassette.This technique leaves a disk of wax protruding from the tissue cassettewith the embedded cells placed at the plane at which a tissue sectioncan be cut using a standard microtome. By placing the filter at theplane of the tissue section, the process of manual embedding iseliminated, and the time required for a technician to find the plane ofthe cells in the embedded block is reduced. Since no other supportingmatrix material is used, the cell fragments are not “diluted” withmatrix, and each tissue section can therefore show a higherconcentration of cells.

The apparatus can be operated manually or be fully automated. A methodis also provided for operating and automating the apparatus of thepresent invention

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a detailed view of a cell block embedding apparatus of thepresent invention;

FIG. 2 is a detailed view of another embodiment of a cell blockembedding apparatus of the present invention;

FIG. 3A is a perspective view of a tissue cassette of the presentinvention;

FIG. 3B is a top-down view of the tissue cassette of FIG. 3A;

FIG. 3C is a bottom-up view of the tissue cassette of FIG. 3A; and

FIG. 4 is a side view of the tissue cassette of FIG. 3A along lines A—A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and apparatus for rapidlypreparing a cell block for sectioning with a microtome. The cell blockembedding apparatus 10 of the present invention is designed as aflow-through system. As shown in detail in FIG. 1, the apparatus 10comprises a cell flow pathway 16 defined by an inflow tube 20 which hasan inlet end 22 configured for placement within a container 12 holding acell sample 14 therein and an outlet end 24 which removably connects toa disposable sample port 26. A solenoid tube clamp 28 can be disposedalong the inflow tube 20 for regulating the flow of fluid through thetube 20. The container 12 can comprise any suitable shape, form, orsize. For example, and as illustrated, the container 12 can be astandard 50 ml disposable centrifuge tube. The cell sample 14 can be insuspension in a physiologic saline solution such as Hanks bufferedsaline, or in a liquid preservative such as 50% aqueous alcohol. Thecell suspension can be vortexed immediately before initiating processingto have the cells suspended. In addition, the cell sample 14 can also bemechanically shaken during the subsequent steps to maintain them in asuspension, for example, by attaching the cell sample container 12 to amechanical shaker (not shown), or by having a disposable mechanicalstirring rod immersed in the cell sample container 12 (not shown).

In fluid communication with the sample port 26 is a disposable reagentport 110 which forms the terminal end of a reagent flow pathway 18 ofthe apparatus 10. The reagent flow pathway 18 is defined by a pluralityof reagent delivery tubes or lines extending from the source of theirrespective reagents and which then converge at the reagent port 110.Embedding reagents 82, 92 and 102 flow to the reagent port 110 throughflow lines 80, 90 and 100, respectively, which are in fluidcommunication with the reagent port 110. Each of the flow lines 80, 90,100 can also include a pump 84, 94, 104 for controlling the delivery ofthe reagent, and a solenoid tube clamp 86, 96, 106 for ensuring anair-tight pathway. In one embodiment, alcohol contained in a reservoir82 can pass through the alcohol flow line 80 xylene contained in areservoir 92 can pass through a xylene flow line 90, and melted paraffincan pass from a heated wax reservoir 102 through a heated wax flow line100 to the reagent port 110. The heated wax flow tube 100 can be formedfrom brass or other heat-conducting materials, while the alcohol andxylene delivery tubes 80, 90 can be formed from a suitable materialwhich is not degraded by the reagents, such as nylon.

While three different reagent flow lines are shown and described, it isunderstood that the reagent flow pathway 18 can comprise any number orcombination of reagent delivery tubes. In addition, a number of otherreagents can also be used in the present invention. For instance, tubesfor delivering formalin or decalcifying acid solutions can also beincluded. Tubes for the delivery of immunohistochemistry reagents canalso be applied with the apparatus 10 of the present invention, therebyenabling protein detection. These tubes can be connected to the reagentport 110 in the same manner as for the alcohol and xylene. Furthermore,it is also possible to have a plurality of reagent delivery tubes fordelivering the same reagent, if so desired.

The sample port 26, cell inflow tube 20 and its removable outlet end 24,and the cell sample container 12 of the present invention are alldisposable, and used for processing only one sample. A baffle can beplaced in the disposable sample port 26 to separate the outlet end 24 ofthe inflow tube 20 from the reagent port 110. This baffle would preventthe cell sample from contaminating the reagent port 110, allowing thereagent port 110 to be used for the subsequent processing of many cellsamples.

As shown in FIG. 1, the sample port 26 can be attached to a removabletissue cassette 30 of the present invention. The tissue cassette 30,illustrated in greater detail in FIGS. 3A—3C, includes a cylindricalport 36 which extends from a top surface 32 through a bottom surface 34of the cassette 30. The cylindrical port 36 creates a flow-throughpathway for the cells and reagents to be delivered. The cylindrical port36 attaches to the sample port 26 from the top surface 32 of thecassette 30 to thereby allow fluid communication between the reagentflow pathway 18 and the tissue cassette 30. Within the cylindrical port36 are thin shelves 44 for anchoring the wax plug that will ultimatelybe formed in the cassette 30 (inside the cylindrical port 36 near thebottom surface 34), though it is understood that other structures suchas mesh, protrusions, ridges, grooves, or surface roughness can also beincluded within the cylindrical port 36 for increased wax adherence. Thetissue cassette 30 is designed to provide sufficient structural rigidityto eliminate the need for wax to fill the entire cassette 30, therebyconserving paraffin. It is understood that tissue cassette 30 should beconstructed from a material which is resistant to degradation fromalcohol, xylene or acids. Exemplary materials for forming the tissuecassette 30 include polypropylene, polypropylene with talc, and nylon.In one embodiment, the tissue cassette 30 has a length in the range ofabout 40-50 mm and a width in the range of about 20-30 mm. The overalldepth of the cassette 30, including the port 36, is in the range ofabout 5-15 mm. As shown in FIG. 4, the cylindrical port 36 of thecassette 30 is approximately 0.5 inches in diameter as measured from thebottom surface 32. These dimensions allow the cylindrical port to fitinto a standard embedding mold, and allow the cassette to be positionedin the holder of a standard microtome for sectioning.

Referring again to FIG. 1, the cylindrical port 36 extending out of thebottom surface 34 of the tissue cassette 30 is removably connected to aflat, ring-shaped, gasket 42. The gasket 42 can be approximately{fraction (1/16)} inch in thickness, and it approximately matches thediameter (0.5 inches) and thickness of the wall of the cylindrical port36 of the tissue cassette 30. The gasket 42 can be composed of a pliablematerial such as Viton™ rubber that allows the gasket 42 to form awater-tight seal with the tissue cassette 30 and resists chemicaldegradation upon exposure to xylene and other embedding reagents. Thegasket 42 should also be sufficiently pliable to allow it to be pulledaway from the hardened wax at the end of the process without deformingthe hardened wax protruding from the tissue cassette.

The gasket 42 is removably connected to a filter 40 that is larger indiameter than the inner diameter of the gasket 42. The filter 40 has apore size in the range of about 6-12 microns in diameter to allowsub-cellular debris to flow through to the filter 40 while trappingsmall-sized individual cells. The size of the pores of the filter can beadapted to different types of samples. For example, a sample known tocontain large numbers of red blood cells and clusters of cells could beprocessed with a filter with 12 micron pore size to allow the individualred blood cells to pass through the filter while trapping the tissuefragments. The filter 40 can comprise a polycarbonate filter (such as,e.g., isopore™ made by Millipore corporation, Billerica, Mass.) whichare smooth and prevent the cells and the wax from sticking to thefilters as they are pulled away. In addition, the gasket 42, tissuecassette 30 and filter 40 can be lightly bonded together so that thesethree components can be quickly loaded onto the apparatus 10.

In one embodiment, the overall thickness of the tissue cassette 30 withthe attached gasket 42 is in the range of approximately 5-20 mm, andpreferably about 10 mm. This low-profile configuration positions thefilter 40 at a location where the lowest surface of a standard tissuemold would be located if the tissue cassette 40 and gasket 42 wereplaced within a standard tissue mold. This tissue cassette 30 and filter40 configuration allows the deposited cells to be automaticallypositioned at approximately the plane at which the microtome blade willcut the cell block, without a need for a tissue mold, since the cellsare embedded within the wax block closest to the plane facing themicrotome cutting blade and farthest away from the attachment site tothe microtome. Since the gasket 42 and tissue cassette 30 can beproduced with accurate dimensions, it is possible to have a microtomepre-set to begin sectioning at exactly the plane where cells arelocated. This feature of the present invention decreases the time andeffort of histologists during the microtome sectioning.

As shown in FIG. 1, the filter 40 rests on a filter support 60. In oneembodiment, an upper surface of the filter support 60 facing the filter40 can be porous in its central portion so that fluids can flow throughthe filter 40 and filter support 60. The filter support 60 can include anon-porous flat ring surrounding the central porous portion. The filter40 can be placed over the filter support 60 to completely cover theporous portion of the filter support 60 and extend somewhat over thenon-porous part of the filter support 60. The gasket 42 can be seated ontop of the outer edge of the filter 40 over the non-porous ring of thefilter support 60. With this configuration, a water-tight seal can beformed between the cylindrical port 36 of the tissue cassette 30 and thefilter support 60, with the filter 40 itself sandwiched therebetween. Inone aspect of the embodiment (not shown), the filter support 60 can havea cylindrical extension to extend over the gasket 42 and bottom end ofthe cylindrical port 36 of the tissue cassette 30. With thisconfiguration, a correct seating of the tissue cassette 30 with respectto the filter support 60, gasket 42, and filter 40 can be assured.

In another aspect, the filter support 60 can include integrated heatingand cooling elements. The filter support 60 can also be configured suchthat heat can be conducted towards or away from the support 60efficiently, in order to maintain the paraffin in a melted state and tospeed up the paraffin hardening process. For instance, a heaterassociated with the filter support 60 can be used to increase the heatsurrounding the support 60 during the embedding process, and decreasethe heat to the support 60 when the embedding is complete and the wax ishardening. Other potential heating and cooling alternatives includemaintaining the entire apparatus 10 in a heated box, or shining infraredlight onto the filter 40.

The filter support 60 is removably connected to a waste container 50 forcapturing the waste fluid during the embedding process, which includesfluid media containing the cell sample, and the excess embeddingreagents. The different effluents can be separated from each other bymechanically moving different waste containers 50 from under the filtersupport 60. Alternatively, the waste container 50 can comprise aplurality of independent receptacles, each with its own solenoid clamp.Such a configuration allows the flow of a particular effluent to beselectively diverted into its respective receptacle by means of thesolenoid clamps. It is contemplated that such a procedure for separatingthe embedding reagents in the effluent would facilitate recycling of theembedding reagents. The apparatus also decreases disposal costs sinceless reagents (e.g., approximately 5 ml per cell block) are usedcompared to other currently available methods of cell block production.

The process of making a cell block can be usefully performed manuallywith this invention, with many of the advantages detailed above,including: more rapid processing speed due to the increased efficiencyof extraction in a flow-through system, decreased use of embeddingreagents, lower risk of mislabeling, elimination of cross-contamination,elimination of the tissue mold step, and standardization of embedding orcell preservation conditions. Because the cell block embedding apparatus10 of the present invention utilizes a flow-through design rather thansimply immersing the cell fragments within embedding solutions, theefficiency of the extractions is dramatically increased, resulting indecreased use of reagents, and extremely rapid processing. In samplesprepared using the apparatus of the present invention, no more than 5 mleach of alcohol, xylene and wax was needed for each cell block produced.The embedding process, when done manually, can be performed in about 10minutes. For a manual process, the cell sample and embedding reagentscould be directly delivered by pipetting to the tissue cassette 30 or tothe sample port 26 under the visual supervision. It is important to loadonly enough cells so that the embedding reagents can flow through thecells on the filter 40. This can be done manually by adding the cellsdropwise while monitoring how fast the meniscus or fluid level of fluiddecreases upon application of a set negative pressure on the waste sideof the filter 40. During operation of the apparatus, when the rate ofmeniscus, or fluid level, descent first perceptibly decreases, there isa sufficiently thick layer of cells for at least twenty 5 micron tissuesections. It has been observed that the rate of fluid level drop isfaster at each of the extraction steps, presumably because the cells aremade progressively more porous as water and fats are removed. It isessential to prevent the meniscus of fluid from ever passing into thefilter 40 during manual performance of the embedding steps; the cellsare distorted if this happens.

The apparatus 10 of the present invention can be configured forautomation as well. Several strategies can be utilized to automate partor all of the cell block production with this invention. For example, itis envisioned that the flow of cell sample and reagents can becontrolled with a combination of positive pressure applied upstream ofthe tissue cassette 30 to push the cell sample and/or embedding reagentsthrough the filter 40, or negative pressure applied on the waste side ofthe tissue cassette 70 to pull the sample and/or embedding reagentsthrough the filter. FIG. 1 illustrates an automated cell block embeddingapparatus 10 in which negative pressure is applied to the wastecontainer 50 by way of a negative pressure tube 70 that can be connectedto a vacuum source. The negative pressure acts to draw the cells fromthe cell sample 14 through the inflow tube 20 and onto the filter 40.The negative pressure also draws the reagents through the filter 40 andover the cells to form the wax cell block. As shown, a pressure gauge 72can be included with the outflow tube for measuring the negativepressure of the system during operation of the apparatus 10. The entiresystem, from the cell sample container 12 and embedding reagentreservoirs 82, 92 and 102, to the waste receptacle 50 can be madewater-tight. In such a water-tight system, the application of a negativepressure at the end of the waste receptacle 70 allows solenoid tubeclamps 86, 96 and 106 to control whether a cell sample 14 is delivered,or whether embedding reagents 82, 92 and 102 are delivered to the sampleport 26 and thereafter to the filter 40.

In a method of operating the cell block embedding apparatus 10, a sampleof cell fragments in an aqueous solution is drawn from a container 12through the filter 40 by applying a vacuum, or negative, pressure fromthe waste container 50 to the inflow tube 20 until the filter poresbecome nearly obstructed by cell fragments. Alcohol is then pulledthrough the filter 40, followed by xylene and melted paraffin. After theparaffin cools, the filter 40 is peeled away from the embedded cells.The gasket 42 between the filter 40 and the tissue cassette 30 is pulledaway from the tissue cassette 30 when the wax hardens, leaving a ring ofwax about 1 mm in thickness protruding from the tissue cassette 30. Thetissue cassette 30 is designed to accommodate the filter 40 such thatthe deposited cells are collected at or very near the plane at which themicrotome blade will cut the cell block for sectioning. The disposablesample port 26 eliminates the risk of cross-contamination between cellblocks since the components that contact the cell sample are disposable.

FIG. 2 illustrates the automated cell block embedding apparatus 10 ofFIG. 1 with an additional positive pressure applied. In this embodiment,a positive pressure tube 74 can be attached to the container 12 havingan airtight lid 76, as shown. The positive pressure tube 74 can beconnected to a positive pressure source for delivering pressure to thecontainer 12, which will then facilitate the movement of cells from thecell suspension in the container 12 through inflow tube 20 and thencethrough the sample port 24 and cassette 30 to the filter 40. Acombination of positive pressure and negative pressures can be used todeliver cell sample and embedding reagents to the filter 40. As shown,the inflow tube 20 include a solenoid tube clamp 28 for ensuring anair-tight pathway. The solenoid 28 serves to keep fluids from runninginto the sample in a positive pressure system, or from having negativepressure pull sample instead of embedding reagents in a negativepressure system.

To automate the system, the inflow of sample and embedding reagents tothe cylindrical port 36 needs to match the outflow into the wastereceptacle 50 such that the meniscus of the liquids passing through thecylindrical port 36 are not pulled into the filter 40, and reagentscannot be allowed to overflow the tissue cassette 30. In a suitablywater-tight system, it may be sufficient to sequentially apply positivepressure to the cell sample container 12 and embedding reagent pumps 84,104, and 104 to force the cell sample and embedding reagentssequentially through the cylindrical port 36 and thereafter through thefilter 40 to effect a useful embedding process.

Alternatively, the amount of cell sample 14 and embedding reagentsdelivered to the cell flow-through pathway 36 can be electronicallymonitored by a variety of techniques and flow rates of sample andembedding reagents can then be appropriately modulated. The meniscus orfluid level can be monitored by existing technologies, or thetechnologies can be specifically adapted to this particular inventionFor example, one mechanism to monitor the meniscus level is to reflectlight at an angle to the meniscus from a laser or light emitting diode.Light sensors can monitor the line of reflectance that change as themeniscus drops. The sensors can be positioned above the tissue cassette,for example, adjacent to the sample port 26, or the sensors can beintegrated into the sample port 26.

Another mechanism to monitor the fluid level is to use a disposable,immersible fiber optic cable, such as provided by ALA ScientificInstruments Inc., Westbury, N.Y. as part of their “level-lock” system.In this system, light leaks out of the cable when it is in contact witha liquid, and the drop in light intensity is proportional to the degreeof immersion. A disposable fiber-optic cable could be incorporated intothe cell flow pathway 16 of the tissue cassette 30 or into the sampleport 24.

Yet another mechanism to monitor the fluid level would be to usecapacitance-type fluid level monitors that detect fluids (includingnon-conducting liquids) because of their different dielectric constantcompared to air. An electrical source and sensor could be positionedjust above the gasket 42.

Still yet another mechanism to monitor the fluid level would be toposition a beam of light horizontally through the lower part of the cellflow pathway immediately above the gasket 42 in a translucent tissuecassette 30. A light sensor placed on the opposite side of the cell flowpathway could detect a change in intensity as the meniscus passedthrough the light beam.

And yet another mechanism to monitor flow rates would be to weigh theeffluent that passes through the filter. Delivery of the sample andembedding reagents could be coupled to the weight of the effluent suchthat the amount of sample or embedding reagents within the cell flowpathway of the tissue cassette is maintained between two levels.

Automated loading of the appropriate amount of cell sample 14 on thefilter 40 could be achieved by a variety of mechanisms. In a suitablewater-tight configuration with solenoid tube clamps 86, 96, 106 closed,application of a modest negative pressure at the waste end of theinvention could deliver the cell sample 14 to the filter 40. A pressuregauge 72 could trigger the release of the negative pressure whennegative pressure is built up to a certain point indicative of theblockage of some of the pores of the filter 40. This mechanism would besimilar to those described in U.S. Pat. Nos. 6,225,125 and 6,010,909.

Alternatively, application to the cell sample container 12 of a shortpulse of positive pressure, as shown in FIG. 2 could force an aliquot ofsample into the cylindrical port 36 of the tissue cassette 30. Theamount of cells delivered could then be estimated by the rate of flowthrough the filter 40. To facilitate this process in which only smallaliquots of cell sample is delivered to the filter 40, it would befeasible to pump in a larger volume of alcohol or saline with each smallaliquot of cell sample delivered. The switching from alcohol to xyleneto wax can be set for a certain volume of reagents to be pumped. Instudies using the apparatus 10 of the present invention, we repeatedlyhave determined that 5 ml of each of these reagents is sufficient forproper embedding. It could also be feasible to monitor the effluent forchanges in refractive index that would indicate that extractions havegone to completion. That is, refractometers can be positioned downstreamof the cell sample in the waste container 50 and the difference in therefractive index between water, alcohol, and xylene could be used todetermine whether all of the reagent (e.g., water, alcohol, xylene,etc.) has been removed.

It is understood that, while three different reagent delivery tubes areshown and described, the reagent flow pathway 18 can comprise any numberand combination of reagent delivery tubes. For example, it may beadvantageous to include a saline reservoir with a flow line to thereagent port 110 to wash away proteins from cell samples 14 contained ina physiologic saline solution. It may also be useful to include an acidreservoir and flow line to the reagent port 110 be able to effect adecalcification of a sample. Excellent quality pre-staining using thepresent apparatus can also be achieved with the present invention. Forexample, the reservoirs could include hematoxylin, eosin and distilledwater to allow a pre-staining of the cell sample, thereby saving timedue to eliminating the need for staining an eventual paraffin section.Eosin is also convenient to include during the alcohol extractionsbecause the eosin makes the cell sample apparent to the histologist whocuts the paraffin section, allowing the histologist to gauge the qualityand quantity of the cells in sections as they are being cut with amicrotome.

By placing the filter at the plane of the tissue section, the process ofmanual embedding is eliminated, and the time required for a technicianto find the plane of the cells in the embedded block is reduced. Sinceno other supporting matrix material is used, the cell fragments are not“diluted” with matrix, and each tissue section can therefore show ahigher concentration of cells. Nevertheless, in some situations it maybe desirable to place the final embedded cell sample into a tissue moldfor re-melting and re-hardening (step 11 of the generic cell-blockprocedure described in the background). This could be done in anautomated fashion. The function of the remelting and re-hardening stepwould be to allow a thin layer of wax to cover the bottom of the cellsto facilitate the “facing in” step of microtome sectioning, whichincreases the number of useable sections that can be cut from a cellblock.

It is understood that the apparatus is configured such that thecomponents are easily removable from one another. This is desirable sothat a technician can quickly and easily replace used or worn componentsin an efficient manner. Further, the sample port 26, cell inflow tube 20and its removable outlet end 24, and the cell sample container 12 of thepresent invention are all disposable. This allows a one-time use of eachof these components for processing one single sample, thereby preventingcontamination while processing subsequent cell samples.

While the cell block embedding apparatus 10 and method of using theapparatus are described herein as a flow-through system, it iscontemplated that the invention could still offer advantages overexisting technology even if the cell flow pathway 16 did not passthrough the tissue cassette 30. For instance, it is envisioned that thecells could still be efficiently and quickly embedded in a temporarychamber overlying the filter 40. Afterwards, the temporary chamber couldbe subsequently remelted in a standard tissue mold, and this remeltingcould be automated.

It is further contemplated that a bar-code reader (not shown) could beincluded with the invention to match the label of the cell sample with alabel on the tissue cassette, and thereby eliminate mislabeling.

It will be understood that the foregoing is only illustrative of theprinciples of the invention, and that various modifications can be madeby those skilled in the art without departing from the scope and spiritof the invention. All references cited herein are expressly incorporatedby reference in their entirety.

1. A flow-through cell block embedding apparatus, comprising: a sampleport; a cell flow pathway defined by an inflow tube that is adapted tobe coupled to the sample port, wherein the inflow tube is effective todeliver cell fragments from a cell sample to the sample port; a tissuecassette in fluid communication with the sample port, the tissue cssetteincluding a removeable and replacable filter such that upon theapplication of pressure, the cell fragments are drawn from the cellsample through the inflow tube to the sample port and deposited onto thefilter; a reagent port in fluid communication with the sample port; anda plurality of reagent delivery tubes adapted to be coupled in fluidcommunication to the reagent port, wherein the plurality of reagenttubes are effective to deliver a plurality of reagents to the reagentport such that upon the application of pressure, the reagents are drawnthrough the reagent delivery tubes to the reagent port to the depositedcell fragments on the filter.
 2. The apparatus of claim 1, wherein thetissue cassette is in fluid communication with the sample port and thereagent port such that the cell fragments are automatically depositednear the plane to be sectioned by a microtome.
 3. The apparatus of claim1, wherein the pressure applied to the reagent flow pathway is anegative pressure.
 4. The apparatus of claim 1, wherein the pressureapplied to the reagent flow pathway is a positive pressure.
 5. Theapparatus of claim 1, wherein the pressure applied to the cell flowpathway is a negative pressure.
 6. The apparatus of claim 1, wherein thepressure applied to the cell flow pathway is a positive pressure.
 7. Theapparatus of claim 1, wherein the reagent flow pathway includes areagent delivery tube for delivering a reagent selected from the groupconsisting of alcohol xylene, hot paraffin, distilled water, saline,acid, hematoxylin, eosin, and immunohistochemistry reagents.
 8. Theapparatus of claim 7, wherein the reagent flow pathway includes a heatedreagent delivery tube for delivering hot paraffin to the sample port. 9.The apparatus of claim 1, wherein each reagent delivery tube furtherincludes a pump for regulating the flow of reagent through the tube. 10.The apparatus of claim 1, wherein each reagent delivery tube furtherincludes a solenoid tube clamp for forming an airtight pathway.
 11. Theapparatus of claim 1, wherein the filter comprises polycarbonate. 12.The apparatus of claim 1, wherein the tissue cassette further includes acylindrical port extending through the cassette configured forattachment to the filter.
 13. The apparatus of claim 12, wherein thecylindrical port is configured for attachment to the sample port. 14.The apparatus of claim 1, further including a waste container forcollecting at least one of the plurality of reagents.
 15. The apparatusof claim 14, wherein the waste container includes a port for connectingto a pressure source.
 16. The apparatus of claim 15, wherein the portfurther includes a pressure gauge.
 17. The apparatus of claim 1, whereinthe sample port is disposable.
 18. The apparatus of claim 1, wherein theapparatus is fully automated.